Fig. 2

DUB knockout library-based screening for DUBs regulating REST protein level by Western blot analysis. (A) Schematic representation of CRISPR/Cas9-based sgRNA library screening for DUBs that alters REST protein levels. Steps 1–2: The designed DUB knockout sgRNA library, consisting of an entire set of genes encoding USPs, was co-transfected with Cas9 into HEK293 cells (day 1). The transfected cells were selected using puromycin (2 µg/mL) and incubated for 3 days at 37 °C in a humidified atmosphere with 5% CO₂ (days 2–5). Step 3: The transfected cells were harvested and lysed in cell lysis buffer and protein was isolated. The protein concentration was estimated by Bradford protein assay. Step 4: Equal concentrations of all DUBKO cell lysates were loaded on SDS-PAGE and screened for DUB candidates regulating endogenous REST levels using western blot analysis. (B) The cell lysates having equal protein concentrations from DUB knockout library were subjected to western blotting to determine the endogenous REST protein level. For each blot, HEK293 cells co-transfected with scrambled sgRNA and Cas9 served as the mock control. GAPDH was used as a loading control. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control for each individual sgRNA (REST/GAPDH) and presented below the blot. (C) The sgRNAs targeting putative DUB candidates and the REST protein level were estimated by western blotting in HEK293 cells. (D) Targeting putative DUB candidates by sgRNAs and its effect on the REST protein level were estimated by western blotting in SH-SY5Y cells. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band for each individual sgRNA (REST/GAPDH) and presented below the blot